System For Propagating Cells

ABSTRACT

Provided is a system and a method for propagating cells, the system comprising a multi well plate and a plate sealing means for sealing at least one well of a multiwell plate, the plate sealing means comprising at least one solid bulge or at least one bulge comprising a solid base, said at least one bulge consisting of a resilient elastomer to securely seal at least one well of the multiwell plate. Also disclosed is the use of such a system for propagating cells and the corresponding method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/566,994, filed Oct. 16, 2017, which claims the priority benefit of UKapplication 1506445.4, filed Apr. 16, 2015, each of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates in general to the field of cell cultures. Moreparticularly, the invention concerns a system for propagating livingcells, and methods for propagating living cells using said system.

BACKGROUND

For analyzing biological mechanisms and for testing toxicity of drugs orchemical compounds, the use of cells and microtissues becomesincreasingly admired. Usually, mammalian cells are cultivated in form ofa two dimensional monolayer of cells adhering to a surface of or withinthe vessel used of cultivating said cell. However, the natural in situenvironment of a cell in a living mammalian organism has athree-dimensional architecture. Thus, cultivating mammalian cells in athree-dimensional architecture leading to the formation of spheroids ormicrotissues provides test systems which more closely resemble themorphological and functional characteristics of a natural environmentthan a monolayer of cells.

Generating spheroids or microtissues is cumbersome, resource-demandingand fiddly. For this reason, spheroids and microtissues are usuallygenerated in facilities possessing the know-how of propagating 3D cellcultures. For use, the spheroids or microtissues are shipped from wherethey are generated to the site of their use, for example, in drugscreening assays. Typically, cells in culture are cooled down to about4° C. or are even frozen for being shipped, regardless of whether thecells are present in a two-dimensional or in a three-dimensionalarchitecture. The cooling or freezing of the cells for and during theirshipment reduces their metabolism and increases their survival rate.However, at their site of use, the cells have to be thawed and/or warmedup to the temperature at which the subsequent assay is performed. Forexample, mammalian cells being propagated at 37° C. will be frozen,shipped in liquid nitrogen or dry ice, and warmed up to 37° C. again insuch a procedure.

Regardless of how gentle the cooling/freezing and thawing/warming isperformed, the viability of the cells within the spheroid/microtissue isaffected as the accuracy and/or reliability of the subsequent assay is.Therefore, there is a need for procedures and means which enablepropagation and shipping of cells, in particular in form of spheroids ormicrotissues, without impairing the cell's viability.

Moreover, it is desired to keep the extent of manipulation of spheroidsand microtissues during propagation prior to an assay to be performed aswell as during the assay as small as possible. Any transfer of spheroidsand microtissues from one culture vessel to another culture vessel mightaffect their integrity and/or functionality. In addition, manipulatingcell cultures always renders the cell cultures prone to contaminationwith unicellular microorganisms such as bacteria or yeasts when the lidof the culture vessel is opened, for example for changing culture media.

Therefore, there is a need for means for culturing cells, in particularin form of microtissues or spheroids, which prevent contamination to theutmost extent.

Mammalian cells may be cultivated in standard SBS/ANSI format multiwellplates which are extensively used in molecular biology laboratories.Multiwell plates are typically rectangular flat plates comprising anarray of wells. The precise dimensions (length×width×height) of amultiwall plate of the ANSI (American National Standard Institute) asrecommended by the SBS (Society for Biomolecular Screening) are 127.76mm×85.48 mm×14.35 mm. Multiwell plates come in a variety of formatswithin said base area but may have a different height. Typical multiwellplates comprise

-   6 wells in a 2×3 array, each of the wells including a volume of 2 ml    to 5 ml;-   12 wells in a 3×4 array, each of the wells including a volume of 2    to 4 ml;-   24 wells in a 4×6 array, each of the wells including a volume of 0.5    ml to 3 ml;-   48 wells in a 6×8 array, each of the wells including a volume of 0.5    to 1.5 ml;-   96 wells in a 8×12 array, each of the wells including a volume of    0.1 to 0.3 ml; or-   384 wells in a 16×24 array, each of the wells including a volume of    0.03 to 0.1 ml.

The multiwell plates are available in different well formats havingeither a flat bottom (F-bottom), a flat bottom with minimal roundededges to the wall (C-bottom), tapered walls (V-bottom) or a bottom inU-shape (U-bottom). Multiwell plates are typically made of polystyreneor polyvinyl chloride. For cell cultures, the surfaces of these productsare modified using an oxygen plasma discharge to make their surfacesmore hydrophilic so that it becomes easier for adherent cells to grow onthe surface which would otherwise be strongly hydrophobic.

Multiwell plates may comprise wells having a circular cross section orwells having a rectangular cross section, preferably a square crosssection.

Multiwell plates may be utilized to generate and propagate spheroids ormicrotissues. For example, multicellular tumor spheroids (MCTS) may begenerated in that equal volumes of a suspension of tumor cells ispipetted into agarose-coated wells of a multiwell plate. At the lowestarea of the agarose-coating of each well only one spheroid forms, andall spheroids within the multiwell plate will have approximately thesame volume. In another approach, microtissues or spheroids may begenerated and propagated in hanging drops, wherein specific multiwellplates are used.

During generating and/or propagating spheroids or microtissues in amultiwell plate, for and during storage of multiwell plates containingspheroids or microtissues, as well as for and during shipping multiwellplates containing spheroids or microtissues, the multiwell plates haveto be covered. Said covering prevents the cell culture in each well fromcontamination and secures integrity of the cell culture. Conventionalpolystyrene lids for multiwell plates are not suitable for shippingmultiwell plates containing spheroids or microtissues at ambienttemperature, because these conventional lids can not prevent spilling ofthe well's content. For avoiding any spill, each of the wells of amultiwell plate bearing cells has to be securely sealed.

For sealing multiwell plates that are used in molecular biologicalmethods such as—for example—Polymerase Chain Reaction (PCR) experiments,a number of ways for sealing the plates are known. For example, a foilor plastic film may be applied across the entire upper surface of theplate. Heat sealable aluminum foils provide an efficient gas and liquidtight seal, but they are tiresome to apply and remove.

Certain adhesive plastic films made of polyethylene plates may providean alternative for sealing multiwell allowing exchange of oxygen, carbondioxide and water vapor. However, these films are tiresome to apply andto remove. In addition, these films are neither autoclavable norreusable. Beyond that, the inventors found that sealing multiwell platesbearing spheroids or microtissues with an adhesive polymer film did notprevent substantial loss of viability when the microtissue-bearingmultiwell plates were kept at a temperature of between 20° C. and 37° C.compared to conventional lids for multiwell plates.

Surprisingly, the inventors found that loss of viability of the cells ofmicrotissues kept at a temperature of between 20° C. and 37° C. issignificantly reduced, if the multiwell plate containing themicrotissues is to sealed with a specifically configured plate sealingmeans made of a resilient elastomer. Moreover, said plate sealing meansimproves handling of microtissue cultures.

SUMMARY OF THE INVENTION

In a first aspect, the invention concerns a system for propagatingcells, wherein said system comprises a multiwell plate and acorresponding plate sealing means for sealing at least one well of saidmultiwell plate.

In a second aspect, the invention concerns a method for propagatingmicrotissues by using the system according to the first aspect.

In a third aspect, the invention concerns the use of the systemaccording to the first aspect for propagating microtissues.

In another aspect, the invention concerns the use of the system in anassay.

In another aspect, the invention concerns a method for investigating theeffect of an analyte on living cells.

In a further aspect, the invention concerns the multiwell plate of thesystem according to the first aspect and its use.

In yet another aspect, the invention concerns the plate sealing means ofthe system according to the first aspect and its use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plane view of an embodiment of the system for propagatingcells.

FIG. 2 shows a cross-sectional view along line C-C of the embodimentshown in FIG. 1.

FIG. 3 shows an enlarged view of the encircled section within thecross-sectional view of the embodiment shown in FIG. 2.

FIG. 4 shows a partial cross-sectional view of a plate sealing meansaccording to prior art and a well of a corresponding multiwell plate.

FIG. 5 shows a partial cross-sectional view of an embodiment of theplate sealing means of the invention and two adjacent wells of acorresponding multiwell plate.

FIG. 6 shows a perspective view of an embodiment of a plate sealingmeans of an embodiment of the system for propagating cells.

FIG. 7 shows a cross-sectional view along line A-A of the embodimentshown in FIG. 6.

FIG. 8 shows a cross-sectional view along line B-B of the embodimentshown in FIG. 6.

FIG. 9 shows an enlarged view of the encircled section within thecross-sectional view of the embodiment shown in FIG. 8.

FIG. 10 shows a cross section through a portion of an embodiment of thesystem.

FIG. 11 shows a cross section through a portion of another embodiment ofthe system.

FIG. 12 shows a cross section through a portion of a further embodimentof the system.

FIG. 13 shows a cross section through a portion of yet anotherembodiment of the system.

FIG. 14 is a cross sectional view of another embodiment of a system forpropagating cells.

FIG. 15A-15C show various embodiments of bulges of a plate sealing meansin cross sectional views.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention will be described with respect to particularembodiments and with reference to certain drawings but the invention isnot limited thereto but only by the claims. The drawings described areonly schematic and are non-limiting. In the drawings, the size of someof the elements may be exaggerated and not drawn on scale forillustrative purposes. The dimensions and the relative dimensions do notcorrespond to actual reductions to practice of the invention.

Furthermore, the terms first, second and the like in the description andin the claims, are used for distinguishing between similar elements andnot necessarily for describing a sequence, either temporally, spatially,in ranking or in any other manner. It is to be understood that the termsso used are interchangeable under appropriate circumstances and that theembodiments of the invention described herein are capable of operationin other sequences than described or illustrated herein.

It is to be noticed that the term “comprising”, used in the claims,should not be interpreted as being restricted to the means listedthereafter; it does not exclude other elements or steps. It is thus tobe interpreted as specifying the presence of the stated features,integers, steps or components as referred to, but does not preclude thepresence or addition of one or more other features, integers, steps orcomponents, or groups thereof. Thus, the scope of the expression “adevice comprising means A and B” should not be limited to devicesconsisting only of components A and B. It means that with respect to thepresent invention, the only relevant components of the device are A andB.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to one of ordinary skill in the art from this disclosure, inone or more embodiments.

Similarly it should be appreciated that in the description of exemplaryembodiments of the invention, various features of the invention aresometimes grouped together in a single embodiment, figure, ordescription thereof for the purpose of streamlining the disclosure andaiding in the understanding of one or more of the various inventiveaspects. This method of disclosure, however, is not to be interpreted asreflecting an intention that the claimed invention requires morefeatures than are expressly recited in each claim. Rather, as thefollowing claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the claimsfollowing the detailed description are hereby expressly incorporatedinto this detailed description, with each claim standing on its own as aseparate embodiment of this invention.

Furthermore, while some embodiments described herein include some butnot other features included in other embodiments, combinations offeatures of different embodiments are meant to be within the scope ofthe invention, and form different embodiments, as would be understood bythose in the art. For example, in the following claims, any of theclaimed embodiments can be used in any combination.

Furthermore, some of the embodiments are described herein as a method orcombination of elements of a method that can be implemented by aprocessor of a computer system or by other means of carrying out thefunction. Thus, a processor with the necessary instructions for carryingout such a method or element of a method forms a means for carrying outthe method or element of a method. Furthermore, an element describedherein of an apparatus embodiment is an example of a means for carryingout the function performed by the element for the purpose of carryingout the invention.

In the description provided herein, numerous specific details are setforth. However, it is understood that embodiments of the invention maybe practiced without these specific details. In other instances,well-known methods, structures and techniques have not been shown indetail in order not to obscure an understanding of this description.

The invention will now be described by a detailed description of severalembodiments of the invention. It is clear that other embodiments of theinvention can be configured according to the knowledge of personsskilled in the art without departing from the true spirit or technicalteaching of the invention, the invention being limited only by the termsof the appended claims.

According to the first aspect, the invention provides a system forpropagating cells, wherein the system comprises a multiwell plate and aplate sealing means for sealing at least one well of the multiwellplate, such that cells within the at least one well of the multiwellplate can be propagated therein when the plate sealing means is appliedto the multiwell plate.

The term “cells” as used herein refers to one or more living cells,either prokaryotic cells or eukaryotic cells. The eukaryotic cells maybe of fungal, plant or animal origin. The term “cells” includes humancells, i.e. cells of human origin. The term “cells” comprises cellsbeing present in form of individual cells, in form of a monolayer ofcells and also cells in form of a spheroid or microtissue.

The term “microtissue” refers to an assembly of cells attached to oneanother. Said assembly of cells may either be present in form of amonolayer of cells or in form of a three dimensional structure. Hence,the term “microtissue” as used herein also comprises spheroids.

The term “propagating” with respect to cells and/or microtissues refersto all aspects of cultivating cells. The term “propagating” as usedherein comprises the cultivation of cells including theirmultiplication, development, proliferation and maturation. Thus,“propagating” also comprises the formation of microtissues. The term“propagating” as used herein also comprises the maintenance, storageand/or shipping of cells and/or microtissues in culture.

In an embodiment, the multiwell plate of the system is a typicalstandard SBS/ANSI format multiwell plate, preferably a multiwell plateas described herein above, more preferably a multiwell plate comprising96 wells in a 8×12 array or 384 wells in a 16×24 array.

In an additional and/or alternative embodiment, at least a portion of atleast one well of the multiwell plate, preferably at least a portion ofall wells of the multiwell plate, is/are provided with an ultra-lowattachment surface. Propagating cells in a well which is provided withan ultra-low attachment surface maintains the cells in a suspended,unattached state. Propagating a spheroid or microtissue in a well thatis provided with an ultra-low attachment surface maintains integrity ofthe spheroid or microtissue, and prevents the spheroid or microtissuefrom undesired attachment-mediated maturation or differentiation,including an undesired alteration in cell division properties.

Providing a well of a multiwell plate with an ultra-low attachmentsurface can be done in various different ways. An example of providing awell with an ultra-low attachment surface is covalently binding ahydrogel layer to the substrate such as polystyrene, said hydrogel beinghydrophilic and neutrally charged. Multiwell plates comprising such anultra-low attachment surface are commercially available from CorningInc. Another example of an ultra-low attachment surface is a surfacemade of a polymer consisting of 2-methacryloyloxyethyl phosphorylcholine(MPC). Such surfaces are available from NOF America Corporation underthe tradename Lipidure®.

Thus in an additional and/or alternative embodiment, at least a portionof at least one well of the multiwell plate, preferably at least aportion of all wells of the multiwell plate comprises a coating layerconsisting of a hydrogel such as agarose, or a coating layer consistingof 2-methacryloyloxyethyl phosphorylcholine (MPC).

In an additional and/or alternative embodiment, at least two neighboringwells of the multiwell plate are in fluid communication. In a preferredembodiment, the wells of a row of wells within the multiwell plate arein fluid communication with each other. For example, in a 96 wellmultiwell plate the 12 wells of up to 8 rows of wells or 8 wells of upto 12 rows of wells can be in fluid communication. In an alternativeembodiment, all wells of a multiwell plate are in fluid communicationwith each other. Preferably, the wells of a row of wells or the wells ofthe multiwell plate are in fluid communication with each other in thateach neighboring wells are in fluid communication.

In a preferred embodiment, the fluid communication between at least twowells of the multiwell plate is established in that at least one channelis provided between said at least two wells.

Preferably, the fluid communication between the two or more wells of themultiwell plate is established by means of at least one channel that isprovided between two wells being in direct fluid communication with eachother. In alterative embodiments, one, two, three, four or even morechannels can be provided between the two wells being in direct fluidcommunication with each other. In embodiments comprising two or morechannels between two wells being in direct fluid communication with eachother, said channels run in parallel.

The diameter of the at least one channel and/or the number of channelsbetween two wells can be chosen to control the flow rate of the fluidfrom one well to the other well. Generally, the lower the number ofchannels and the smaller its/their diameter, the smaller the flow rateof the medium that may migrate from one well to the next well.

Providing a fluid communication between at least two wells of themultiwell plate permits the system's use for assays wherein the effectof a metabolite of product produced by the cells in one well on thecells with the other well can be determined without any need of openingthe sealing of the wells and can also avoid the need of aspiratingculture medium from wells bearing cells.

The plate sealing means comprises at least one bulge consisting of aresilient elastomer, wherein the bulge is configured to securely fitinto a well of the multiwell plate. The term “resilient” means that thematerial is able to return to its original shape after being pulled,stretched, pressed or bent without permanent deformation or rupture.Thus, the bulge fits into the opening of the well, and due to itsresilience securely seals the well. The term “securely sealing” meansthat the bulge does not drop out of the well when the system is turnedupside down, and that no fluid inside the well leaks out of the sealedwell when the system is turned upside down.

In an additional and/or alternative embodiment, the at least one bulgeis a solid bulge. The at least one bulge may be configured as a frustrumof a cone or as a frustrum of an inverse cone. In an additional and/oralternative configuration, the at least one bulge comprises a concavebottom for providing an obstruse angle between the wall of the well andthe bulge when the bulge is appropriately inserted into said well. Theterm “bottom” with respect to a bulge refers to the distal end of thebulge, i.e. the end of the bulge being opposite of its base. In anadditional and/or alternative configuration, the at least one bulgecomprises a hollowed out bulge for improved flexibility and fit of saidat least one bulge into a corresponding well. The hollowing out of thebulge provides a flexible edging of the bulge which improves fit of theat least one bulge in the well and sealing of the well. In yet anadditional and/or alternative embodiment, the at least one bulgecomprises at least one flap. Preferably, the at least one bulgecomprises a single flap or a double flap. Said at least one flap iscircumferentially arranged at the outer edge of the distal portion ofthe at least one bulge. The distal portion of the at least one bulge isunderstood to be the portion opposite of the bulge's base.Notwithstanding, the configurations comprising a concave bottom, ahollow out and/or at least one flap comprise a base portion being solidin its entire cross section.

In an additional and/or alternative embodiment, the resilient elastomeris a silicone rubber.

A preferred silicone rubber has a hardness of Shore A in the range of 55to 65, measured according to DIN 53505.

A preferred silicone rubber has a specific gravity of between about 1.13and 1.17 g/cm3, measured according to DIN 53479.

A preferred silicone rubber has a tensile strength of about 7 MPa,measured according to DIN 53504 S2.

A preferred silicone rubber has an elongation of about 350%, measuredaccording to DIN 53504 S2.

In an additional and/or alternative embodiment, the resilient elastomeris selected from the group consisting of silicone rubber, preferablydimethylsilicone rubber vinyl methyl siloxane, and phenyl vinyl methylsiloxane, fluorosilicone rubber, and nitrile rubber and natural rubber.

In an additional and/or alternative embodiment, the resilient elastomeris gas permeable. This is, that gas such as—for example—air, oxygen andcarbon dioxide—can permeate through the silicone rubber. In anadditional and/or alternative embodiment, of the gas permeable resilientelastomer has an oxygen permeability of more than 1*10⁹,cm³*cm/(s*cm²*cmHg).

In an additional and/or alternative embodiment, the plate sealing meansis antistatic. This is, the silicone rubber material of the platesealing means reduces or even eliminates static electricity.

In an additional and/or alternative embodiment, the plate sealing meansis autoclavable. That is, the plate sealing means can be sterilizedwithout being damaged or impaired in its functionality as describedherein by subjecting them to high pressure saturated steam at 121° C.for around 15 to 20 minutes.

In an additional and/or alternative embodiment, the plate sealing meansis reusable. This is, the plate sealing means can be used multiple timesfor sealing a multiwell plate without being damaged or impaired in itsfunctionality.

In an additional and/or alternative embodiment, the plate sealing meanscomprises at least one bulge comprising a resealable septum. This is,the plate sealing means may be punctured by means of a 18 gauge needleand reseals when the 18 gauge needle is pulled out of the plate sealingmeans.

The properties such as gas-permeability is not significantly alteredupon puncturing.

In an additional and/or alternative embodiment, the plate sealing meansis non-adhesive. This is, the plate sealing means does not have a tack.

In an additional and/or alternative embodiment, the at least one bulgeof the plate sealing means comprises a rim at its base, i.e. where thebulge protrudes from the second face. The rim of the at least one bulgeis circumferential. The rim of the at least one bulge is configured notto fit into a well of a corresponding multiwell plate, but to sit on topof the well next to the well's aperture.

The rim prevents the second face of the plate sealing means from gettinginto contact with the upper face of the multiwell plate. Thisconfiguration permits easier removal of the plate sealing means from themultiwell plate compared to an embodiment without such rims.

The at least one bulge may have a circular cross section, a rectangularcross section of a square cross section. The sides of the bulge areconfigured to narrow the cross section from the base of the bulgetowards the tip of the bulge. In an embodiment, the opposite sides of abulge include an angle of 30° which corresponds to an angle of 15°between the longitudinal axis of the bulge and the flange.

An angle of approximately 30° between the sides of a bulge permits easyinsertion and removal of the bulge into/from a well of a correspondingmultiwell plate, but secures tight and snugly fitting of the know withinthe well.

In an additional and/or alternative embodiment, the plate sealing meansis present in form of a resilient mat made of a gas-permeable,antistatic silicone rubber. The plate sealing means of this embodimentcomprises a first face and a second face. The first face constitutes theupper face of the plate sealing means when the plate sealing means is inuse, i.e. applied to a multiwell plate. Thus, the first face of theplate sealing means faces away from the multiwell plate when the platesealing means is applied to a multiwell plate. The second face of theplate sealing means is at the opposite side of the first face of theplate sealing means and constitutes the lower face, i.e. the face of theplate sealing means facing the multiwell plate when the plate sealingmeans is in use, i.e. applied to a multiwell plate.

In an embodiment, the first face has a flat or even surfacesubstantially free from dimples. In an additional and/or alternativeembodiment, the first face has labelling means for better identifyingthose areas of the first face being aligned with the wells of amultiwell plate when the plate sealing means is applied to a multiwellplate. The labelling means may be an embossed, imprinted or engravedstructure. The labelling means may comprise letters, numbers orcombinations of letters and numbers unique for each well, rims or wallsindicating the position of each well, wherein the labelling means arearranged on the first face of the plate sealing means at positionscorresponding to the wells of the multiwell plate, i.e. opposite ofbulges protruding from the second face of the plate sealing means.

The plate sealing means in form of the resilient mat comprises at leastone bulge protruding from the second face of the plate sealing means.Preferably the plate sealing means comprises an array of bulgesprotruding from the second face of the plate sealing means, wherein thearray of bulges matches the array of wells of a corresponding multiwellplate, i.e. of a multiwell plate said plate sealing means is configuredto seal. The at least one bulge, preferably each bulge of the array ofbulges, is/are sized and shaped to sit firmly in a well of thecorresponding multiwell plate.

The at least one bulge or the bulges or the array of bulges are solidelements of the plate sealing means protruding from the second face ofthe plate sealing means or elements comprising a solid base portion.That is, the bulges are solid or comprise a solid base, and they consistof the silicone rubber, the plate sealing means are made of such thatthe at least one bulge or the bulges are integral elements of the platesealing means such that the thickness of the rubber material isincreased in the area of the bulges compared to the area between twobulges. The bulges are not configured as dimples which are typicallygenerated in that the rubber film is deformed such that the thickness ofthe rubber material in the area of the dimple is essentially the same asoutside of the dimple.

The bulges provide a secure sealing of the wells of a correspondingmultiwell plate when the plate sealing means is applied to the multiwellplate, thus preventing any liquids from escaping from or entering thewell that is sealed. It is believed that the tight sealing of the wellsis based on the fact that the bulge is solid or comprises a solid baserather than being a dimple. The restoring force of the resilient bulgewhen inserted into the well of the multiwell plate appears to be higherthan the restoring force of a dimple having the same shape and size.Therefore, it is believed that the better sealing of a bulge compared toa dimple accounts for the improvement in cell viability.

Additional configurations of the bulge further improve their efficacy insealing a well and thereby securing propagation of cells within thewell. Such additional configurations are—for example—a concave bottom ofthe bilge, a hollow out of the bulge and/or at least one circumferentialflap at the distal portion of the bulge.

The plate sealing means comprising solid bulges or bulges comprising asolid base is made of a resilient elastomer. Thus, a flexible mat isprovided that can be easily applied to a multiwell plate, easily removedfrom the multiwell plate and easily re-applied to the same or anothermultiwell plate, and simultaneously provide a tight sealing of the wellsof a multiwell plate.

In an embodiment, the plate sealing means covers the entire surface ofthe multiwell plate. In an alternative embodiment, the plate sealingmeans does not cover the entire surface of the multiwell plate, but onlya portion of the surface.

The plate sealing means comprises a resilient silicone mat which coversessentially the entire upper surface of a standard 96-well plate, andcomprises 96 bulges such that every well of a standard 96 well plate canbe sealed using this embodiment of the plate sealing means.

In an embodiment, the plate sealing means comprises a circumferentialbrim. The brim extends perpendicular to the plane of the mat and in thedirection of the bulges. Thus when mounted to a multiwell plate the brimof the plate sealing means encompasses the sides of the multiwell plate.Thus the embodiment of the plate sealing means has the form aconventional polystyrene lid for multiwell plates, but is resilient.

The bulges preferably have a circular cross section for securely fittinginto wells having a circular cross section too. It is understood thatthe bulges of the plate sealing means may be configured as describedherein before with respect to the bulges.

The term “multiwell plate” as used herein refers to rectangular flatplates comprising an array of wells, preferably having dimensions asrecommended by the Society for Biomolecular Screening as set forthherein above.

The term “corresponding multiwell plate” as used herein refers to amultiwell plate which has the same number of wells as the plate sealingmeans has bulge, wherein the wells of the multiwall plate are present inthe same array as the bulges of the plate sealing means are, and/orwherein the wells have the same cross section as the bulges of the platesealing means have, and where the diameter of the well is identical tothe diameter of the bulge at about half of its height. The term“corresponding multiwell plate” shall indicate that the plate sealingmeans is a plate sealing means configured for sealing the wells of aspecific multiwell plate.

The term “cultivating” as used herein comprises maintaining orpropagating cells in vitro, wherein the term “maintaining” comprisesstorage of cells at lower temperatures, for example at about 4° C., oreven frozen, for example at about −20° C., about −80° C. or even atabout −180° C. The term “cultivating” comprises proliferation of cellsin vitro as well as the maintenance of cells in vitro, i.e. underconditions where the cells multiply, multiply at a reduced speed, or donot multiply.

The term “shipping” as used herein refers to freight transport, i.e. thephysical process of transporting commodities and merchandise goods andcargo including living organs, tissues, microtissues, spheroids andcells. The term “shipping” comprises transport by sea, by land or air.

In an embodiment the plate sealing means is made of a resilientelastomer, the resilient elastomer is preferably selected from the groupconsisting of silicone rubber, preferably dimethylsilicone rubber vinylmethyl siloxane, and phenyl vinyl methyl siloxane, fluorosiliconerubber, and nitrile rubber and natural rubber.

In an embodiment, the resilient elastomer of the plate sealing means isgas permeable. This is, that gas such as—for example—air, oxygen andcarbon dioxide—can permeate through the elastomer. Preferably, theresilient elastomer has an oxygen permeability of more than 1*10⁹,cm³*cm/(s*cm²*cm Hg).

A preferred silicone rubber has a hardness of Shore A in the range of 55to 65, measured according to DIN 53505.

A preferred silicone rubber has a specific gravity of between about 1.13and 1.17 g/cm3, measured according to DIN 53479.

A preferred silicone rubber has a tensile strength of about 7 MPa,measured according to DIN 53504 S2.

A preferred silicone rubber has an elongation of about 350%, measuredaccording to DIN 53504 S2.

The plate sealing means is antistatic. This is, the silicone rubbermaterial of the plate sealing means reduces or even eliminates staticelectricity.

The plate sealing means is autoclavable. That is, the plate sealingmeans can be sterilized without being damaged or impaired in itsfunctionality as described herein by subjecting them to high pressuresaturated steam at 121° C. for around 15 to 20 minutes.

The plate sealing means is reusable. This is, the plate sealing meanscan be used multiple times for sealing a multiwell plate without beingdamaged or impaired in its functionality.

In an additional embodiment, the plate sealing means comprises at leastone bulge comprising a resealable septum. This is, the plate sealingmeans may be punctured by means of a 18 gauge needle and reseals whenthe 18 gauge needle is pulled out of the plate sealing means. Theproperties such as gas-permeability is not significantly altered uponpuncturing.

The plate sealing means is non-adhesive. This is, the plate sealingmeans does not have a tack.

In an additional embodiment, the at least one bulge of the plate sealingmeans comprises a rim at its base, i.e. where the bulge protrudes fromthe second face. The rim of the at least one bulge is circumferential.The rim of the at least one bulge is configured not to fit into a wellof a corresponding multiwell plate, but to sit on top of the well nextto the well's aperture.

The rim prevents the second face of the plate sealing means from gettinginto contact with the upper face of the multiwell plate. Thisconfiguration permits easier removal of the plate sealing means from themultiwell plate compared to an embodiment without such rims.

The at least one bulge may have a circular cross section, a rectangularcross section of a square cross section. The sides of the bulge areconfigured to narrow the cross section from the base of the bulgetowards the tip of the bulge. In an embodiment, the opposite sides of abulge include an angle of 30° which corresponds to an angle of 15°between the longitudinal axis of the bulge and the flange.

An angle of approximately 30° between the sides of a bulge permits easyinsertion and removal of the bulge into/from a well of a correspondingmultiwell plate, but secures tight and snugly fitting of the know withinthe well.

In an embodiment, the plate sealing means comprises a circumferentialbrim. The brim extends perpendicular to the plane of the mat and in thedirection of the bulges. Thus when mounted to a multiwell plate the brimof the plate sealing means encompasses the sides of the multiwell plate.Thus the embodiment of the plate sealing means has the form aconventional polystyrene lid for multiwell plates, but is resilient.

The plate sealing means may additionally comprise posts or pins fittingrespectively into holes other than the wells within a multiwell plate.The posts are shaped such that they fit into the holes and contact thewalls of the holes. The posts/holes are further means preventing theplate sealing means from moving laterally with respect to the multiwellplate, and prevent misalignment of the plate sealing means, inparticular when the plate sealing means is only partially removed fromthe multiwell plate, for instance when access to individual wells of themultiwell plate is required.

In an embodiment, the plate sealing means comprise at least one inletopening and at least one outlet opening. In a preferred embodiment, theplate sealing means comprises an inlet opening and an outlet opening foreach row of wells within the multiwell plate. Preferably, each of theinlet openings of the plate sealing means corresponds to the first wellof the row of wells, whereas the outlet well corresponds to the lastwell of the row of wells. Said at least one inlet opening and said atleast one outlet opening are holes or through bores in the plate sealingmeans such that the corresponding well of the multiwell plate is notsealed by the plate sealing means when the plate sealing means ismounted to the multiwell plate.

The inlet opening permits adding or supplementing culture medium to theat least one well. The outlet opening permits removal or aspiration ofculture medium from the at least one well. In combination with theembodiment of the multiwell plate comprising at least two wells being influid connection, mounting of the plate sealing means comprising atleast one inlet opening and one outlet opening permits supplementing allwells with culture medium that are in direct or indirect fluidconnection with inlet well.

In a further embodiment, the at least one inlet opening and/or the atleast one outlet opening of the plate sealing means are configured asfunnel or chimney. “Funnel” refers to a structure having a diameternarrowing towards the point of connection to the plane of the siliconemat, at least along of at least a portion of its longitudinal direction.“Chimney” refers to a pipe or tube like structure having a constantdiameter along its longitudinal direction.

In another embodiment, the plate sealing means is configured in in formof a substantially planar resilient mat without bulges. In thisembodiment, the plate sealing means comprises a rigid frame, preferablymade of hard plastics such as polystyrene, and is configured to be usedas a lid for a corresponding multiwell plate. Said rigid frame comprisesa plate comprising bores, wherein said bores align to the wells of acorresponding multiwell plate. Said plate comprises an outer face and aninner face. The outer face faces away from the multiwell plate, whereasthe inner face faces towards the multiwell plate upon mounting the platesealing means to the multiwell plate. The substantially planar resilientmat is provided on the inner face of the plate.

In an additional and/or alternative embodiment, the system comprises aquick lock system for securing the rigid frame to the multiwell plate.In an embodiment, the quick lock system comprises at least two lockinglatches at opposite ends of the frame. Said latches engage with groovesor slots or trenches at the corresponding positions at the outercircumference of the corresponding multiwell plate. By forcing the frameonto the multiwell plate, the latches engage and thereby not onlysecures the frame onto the multiwell plate, but simultaneously sealingthe wells of the multiwell plate.

In a preferred embodiment of the multiwell plate for being sealed by theplate sealing means of the latter embodiment, each well comprises acircumferential edge at its outlet opening. Said circumferential edgeprotruding from the upper face of the multiwell plate in perpendiculardirection thereto. Said circumferential rim is pressed into the planarresilient mat when the plate sealing means is mounted to the multiwellplate, thereby providing the secure sealing if the well.

The substantially planar resilient mat is made of a resilient elastomeras described herein before with respect to the bulges.

Referring to FIGS. 1 to 3 an embodiment of the system (1) forpropagating microtissues is displayed. FIG. 1 shows a perspective viewon top of the system (1). The embodiment comprises a 96-well multiwellplate (10) comprises a plurality of wells (11) being arranged in a 12×8array. The multiwell plate (10) is covered with an embodiment of a platesealing means (20, not shown in FIG. 1).

The embodiment of the multiwell plate (10) comprises three positioningmeans (12, 13, 14) in form of apertures. The three positioning means(12, 13, 14) are arranged such that a lid or a plate sealing means (20)comprising corresponding protuberances (22, 23, 24) which are configuredfor engaging said positioning means (12, 13, 14) can be applied to themultiwell plate (10) in one specific orientation only.

The plate sealing means (20) comprises a plurality of bulges (21)protruding from one face of the plate sealing means. The plurality onbulges (21) are present in the same array as the wells of thecorresponding multiwell plate. In the embodiment shown in FIGS. 1 to 3,the plurality of bulges (21) are arranged in a 12×8 array.

The presence of the positioning means (12, 13, 14) prevents a platesealing means (20) comprising a symmetrical array of bulges (21) to theapplied to the multiwell plate (10) in an inadvertent orientation.Thereby avoiding contamination.

In addition, the embodiment of the plate sealing means (20) shown inFIG. 2 comprises a circumferential brim (25) at the outer border of theplate sealing means (20). The brim (25) extends the sides of the platesealing means (20) over the upper surface of the multiwell plate (10)when the plate sealing means (20) is appropriately mounted to themultiwell plate (10).

FIG. 3 illustrates how a bulge (21) of the plate sealing means (20)snugly fits into the corresponding well (11) of a correspondingmultiwell plate (10) providing a tight seal of the well (11) due to itssize and resilience. The bulge (21) is configured to leave a volume forcells, culture medium and/or air in the well (11).

FIG. 4 shows a cross-sectional view of a part of a commerciallyavailable plate sealing means (30). The view represent a single well ofthe multiwell plate and the corresponding section of a commerciallyavailable plate sealing means (30). The commercially available platesealing means (30) consists of a film that has been subjected to anembossing process such that dimples (31) are generated. Said dimples(31) are configured to fit into a well (11) of a corresponding multiwellplate (10). The thickness of the film in the areas of the dimples is thesame as in the areas next to the dimples.

Referring to FIG. 5 showing a cross-sectional view of a part of anembodiment of a system according to the invention. The part representstwo neighboring wells (11) of a multiwell plate (10), and thecorresponding section of a corresponding plate sealing means (20). Thefigure illustrates that the plate sealing means (20 comprises bulges(21) which are solid elements of the plate sealing means (20) were thematerial of the plate sealing means (20) is much thicker than in theregion between the bulges (21).

The bulges (21) of the embodiment of the plate sealing means (20) asshown in FIG. 5 comprise a rim (26) at the base of the bulges (21), i.e.where the bulges (21) protrude from the plane of the plate sealing means(20). Upon sealing of a well (11) of a corresponding multiwell plate(10), the bulges (21) fit snugly into the open end of the well (11) suchthat the rim (26) resides on the upper face (16) of the multiwell plate(10). The rim (26) of each bulge (21) is a circumferential element. Thepresence of said rims (26) at each bulge (21) renders removal of theplate sealing means (20) from the corresponding multiwell plate (10)after the plate sealing means (20) being mounted to the multiwell plate(10) much easier, thereby preventing undesired shaking which mightaffect formation and/or integrity of a microtissue within a well of themultiwell plate (10) and/or might lead to undesired spillover of culturemedium from one well to the other.

Referring to FIGS. 6 to 9 an embodiment of the plate sealing means (20)is displayed. FIG. 6 shows a perspective view on top of the second face(27) of the plate sealing means (20). The embodiment of the platesealing means (20) as shown comprises an array of solid bulges (21)protruding from the second face (27). The plurality of solid bulges (21)are arranged in an 8×12 array corresponding to the array of wells of atypical 96-well plate.

FIG. 7 illustrates a longitudinal section along line A-A of the platesealing means (20) of FIG. 6, whereas FIG. 8 illustrates a longitudinalsection along line B-B of the plate sealing means (20) of FIG. 6.

FIG. 9 is an enlarged view of portion II encircled in FIG. 8. FIG. 9emphasis a solid bulge (21) of the plate sealing means (20) whichprotrudes from the second face (27) of the plate sealing means.

The solid bulges (21) protruding from the second face (27) of the platesealing means (20) preferably have a height of about 1.5 times thethickness of the mat, i.e. the distance measured from the first face(28) to the second face (27) of the mat. The bulges (21) comprise a basewhere they merge with the second face (27) of the plate sealing means(20), and a tip. The sides (29) of each bulge (21) are tapered such thatthe diameter of the bulge (21) at its tip is smaller than the diameterat the base of the bulge (21). The flanges (29) on opposite sides of thesolid bulge (21) include an angle (a) of about 30° such that the bulgecan be easily inserted into a well of a corresponding multiwell plate,fits snuggly into the well, and provides a tight seal of the well. Thesides are preferably tapered in that they include an angle (7) of about30°.

FIG. 10 illustrates a portion of a further embodiment of a system,wherein the plate sealing means (20) comprises at least one solid bulge(21) for sealing the corresponding well (11) of a correspondingmultiwell plate (10). The bulge (21) comprises a self-sealing septumsuch that, for example, an injection needle (40) can be poked throughthe self-sealing septum of the bulge (21). Using a syringe or othersuitable means, culture medium can be aspirated trough the injectionneedle (40) from the well (11) and/or added thereto without the need ofremoving the entire plate sealing means (20) from the multiwell plate(10). Upon removal of the injection needle (40), the septum seals itselfsuch that the plate sealing means (20) still provides a tight seal ofthe well (11).

Also shown in FIG. 10 is an embodiment comprising a specificallydesigned well (11) for propagating microtissues (2). Said well (11)comprises a microtissue culture compartment (15) which is present at thebottom of the well (11). Said microtissue culture compartment (15) is asmall partition in the middle of the bottom of the well which is influid connection therewith and thus constitutes an integral part of thewell.

FIG. 11 illustrates a portion of another embodiment, wherein the platesealing means (20) comprising a bulge (21) with two self-sealing septa.This embodiment permits inserting an inlet conduit (41) and an outletconduit (42) through the plate sealing means (20) into the well (11).This configuration permits providing a constant flow of culture mediumthrough the well (11).

FIG. 12 is a schematic representation of another embodiment of thesystem (1) for propagating microtissues. The multiwell plate (10) isconfigured such that the neighboring wells (11, 11′, 11″) of one row ofwells of the multiwell plate (10) are in fluid connection with oneanother by means of at least one channel (17) connecting the wells.

In addition, the embodiment comprises a plate sealing means (20),comprising an inlet opening (210) and an outlet opening (220). The inletopening (210) is placed instead of the first bulge of a row of bulges,whereas the outlet opening (220) is placed instead of the last bulge ofthe row of bulges.

The inlet opening (210) and/or the outlet opening (220) may beconfigured as through holes in the silicone mat. In an alternativeembodiment the inlet opening (210) and/or the outlet opening (220) maybe configured as a funnel or chimney which comprises a wall (211, 221)extending substantially upright from the first face (28) of the platesealing means (20) when mounted to the multiwell plate (10).

The open end of the inlet opening (210) and the open end of the outletopening may be sealed by means of a plug (43, 45). The plug may be madeof a plastic material such as a silicone rubber. In an embodiment, theplug (43) may comprise a microporous section (44). Alternatively, theplug (45) may consist of a microporous material. Sealing the inletopening and/or the outlet opening of the plate sealing means with a plugconsisting of or to comprising a microporous material improves theexchange of gaseous fluids in the system (1).

FIG. 13 shows a cross section through a portion of an embodiment of thesystem comprising a multiwell plate (10) and a plate sealing means (20).The multiwell plate comprises a plurality of wells being in fluidcommunication with one another. Neighboring wells (11) are put in fluidcommunication by means of one or more channels (17) extending from onewell (11) to the next well in a row of wells.

In a preferred embodiment, the one or more channels connectingneighboring wells is/are not disposed at the lower end of the well, butat a distance in height from the bottom of the wells.

The embodiments shown in FIGS. 12 and 13 allow to supply culture mediumthrough the inlet opening of the plate sealing means mounted to thecorresponding multiwell plate without the need of removing the platesealing means (20) from the multiwell plate (10). The culture mediumsupplied to the first well, i.e. the well underneath the inlet opening(210) will flow through the channels connecting the wells with oneanother such that all wells being in fluid communication with each otherare supplied with culture medium.

The embodiment further provides the opportunity of providing a constantflow of culture medium through the wells being in fluid communication ifa constant supply of culture medium through the inlet opening (210) anda constant removal of culture medium through the outlet opening (220) isprovided. The constant flow of culture medium may be obtained byutilizing hydrostatic forces in that the level of culture medium in thefunnel of the inlet opening is kept higher than the level of culturemedium at the outlet opening. The height difference thus provides ahydrostatic force which forces the culture medium flowing through thechannels from one well to the next well towards the last well which isthe well underneath the outlet opening. The velocity of medium flowthrough from one well to the next well can also be determined by thediameter and/or number of channels connecting said wells.

Referring to FIG. 14, another embodiment of the system is shown. Thesystem (1) of this embodiment comprises a multiwell plate (310) and aplate sealing means (320). The multiwell plate (310) comprises wells(311, 311′) including microtissue compartments (315, 315′). The wells(311, 311′) are in fluid communication due to the channel (317) which isprovided between said wells (311, 311′). In addition, each of the wells(311, 311′) comprises a circumferential edge (325, 325′) protruding fromthe upper face of the multiwell plate (310). The multiwell plate furthercomprises grooves (312, 312′).

The embodiment further comprises a plate sealing means (320) which isconfigured as a lid comprising a plate (326) and a circumferential brim(322). Said brim (322) comprises latches (323, 323′) at opposite sidesof the lid for engaging in the grooves (312, 312′) upon correct mountingof the plate sealing means (320) to the multiwell plate (310). The platesealing means (320) further comprises a planar mat (321) made of aresilient elastomer. Said planar mat (321) may be attached to the plate(326). The plate further comprises through bores (324, 324′) which alignwith the wells (315, 315′) of the multiwell plate upon being mountedthereto.

In an embodiment of the first aspect, the system further comprises meansfor controlling the temperature within the wells of the multiwell plate.

The means for controlling the temperature are configured to control thetemperature such that the temperature within the wells of the multiwellplate is a temperature of between 25° C. and 37° C.

In an alternative and/or additional embodiment, the means forcontrolling the temperature within the wells of the multiwell plate areconfigured to control the temperature during shipping of the multiwellplate being sealed with a plate sealing means according to the firstaspect.

In another and/or alternative embodiment of the first aspect, the systemfurther comprises a temperature data logger. The temperature datalogger, also called temperature monitor, is a portable measurementinstrument that is capable of autonomously recording the temperatureover a defined period of time. The digital data being recorded can beretrieved, viewed and evaluated. The temperature data logger is used tomonitor the temperature of the cells being propagated in that theambient temperature of the cell culture is measured. Using thetemperature data logger is advantageous when the cells or microtissuesare shipped. The temperature data logger is included in the containerbearing the cell culture to be shipped within at least one sealedmultiwell plate. The temperature data logger is preferably placed inclose proximity to the at least one multiwell plate including the cellsor microtissues. The temperature data logger monitors the temperatureand alterations in temperature within the container and thus thetemperature said cells or microtissues are exposed to. This temperaturemonitoring permits use of only those cells or microtissues for any assaythat were not exposed to undesired temperatures or inadequatetemperatures.

Referring to FIG. 15 a to c various embodiments of bulges are shownschematically in cross-sectional views. FIG. 15 a shows embodiments (Ito IV) configures as a frustrum of a cone, wherein the bulges comprise aconcave bottom (101, 102, 103, 104). Embodiments III and IV areconfigured as inverse frustrum of a cone. FIG. 15 b shows embodiments (Vto VIII) wherein the bulges comprise a hollow out (111, 112, 113, 114).The bulges comprise a solid portion at their base (121, 122, 123, 124)and a circumferential rim (131, 132, 133, 134) at their distal portion.FIG. 15 c shows embodiments of bulges comprising a single flap(embodiments IX and X) of a double flap (embodiment XI). The singleflaps (141, 142) and the double flap consisting of two flaps (143 and144) are configured at the outer wall of the bulge where they extendoutwards and circumferentially from the circumferential rim of thebulge. In the embodiments shown, the flaps (141, 142 and 144) arearranged at the distal end of the bulge. The bulges as shown comprise asolid base, a hollow out and a circumferential rim as exemplified withrespect to embodiment IX, wherein the solid base (151) extendsperpendicular from the second face (27) of the plate sealing means (20).The bulge comprises a hollow out (161) providing a circumferential rim(171) which further extends from the plate sealing means.

Upon inserting a bulge comprising a concave bottom, a hollow out and/orat least one rim into a corresponding well, the distal portion of saidbulge can better snuggle to the inner lining of the wall of the well andthereby provide an even better sealing of the well than a solid bulge.

It is understood that the dimensions of a bulge may vary upon thedimension of the well to be sealed, and that bulges of different heightsmay be provided.

According to the second aspect, the invention provides a method ofpropagating cells, wherein cells are provided and propagated in at leastone well of a multiwell plate according to the system, said at least onewell of the multiwell plate being sealed with a plate sealing meansaccording of the system.

The method comprises the steps of:

-   -   providing a multiwell plate as described herein before;    -   introducing a suspension comprising at least one cell into at        least one well of the multiwell plate;    -   sealing at least the well of the multiwell plate bearing the        suspension containing at least one cell with a plate sealing        means as described herein before.

In an embodiment of the method, said cells are eukaryotic cells. In afurther and/or additional embodiment, the cells are mammalian cells. Inyet a further and/or alternative embodiment, the cells are human cells.

In an embodiment of the method, the cells are propagated, propagatingthe cells at a temperature of between 4° C. and 37° C., preferably at atemperature of between 18° C. and 37° C., more preferably at atemperature of between 25° C. and 37° C., and most preferably at atemperature of between 29° C. and 37° C. for a period of time.Alternatively at a temperature of between 18° C. and 27° C., preferablyat a temperature of between 21° C. and 25° C.

The temperature for cultivating and/or shipping cells is preferablymaintained constant by utilizing suitable means of controlling thetemperature.

In an embodiment of the method, the propagation comprises providing anexchange of culture medium. Said exchange of culture medium may beperformed continuously or discontinuously.

In an embodiment, wherein the culture medium is discontinuouslyexchanged in individual wells of the system, an injection needle ispoked through the plate sealing means, preferably through a self-sealingseptum of the bulge sealing said well, and inserted into the well. Ifalready present, the culture medium in the well is aspirated through theneedle, and fresh culture medium is supplied to the well through theneedle.

In an embodiment wherein the culture medium is continuously exchanged inindividual wells of the system, an inlet conduit and an outlet conduitare inserted through the plate sealing means, preferably through one ormore self-sealing septa of the bulge sealing said well, into the well. Acontinuous flow of culture medium through the well is then provided bysupplying the culture medium through the inlet conduit and by removingculture medium through the outlet conduit.

In an embodiment wherein a system comprising one or more wells being influid communication with each other, a continuous exchange of culturemedium can be provided in that fresh culture medium is supplied throughthe inlet opening of the plate sealing means while being mounted to thecorresponding multiwell plate, and removing culture medium through theoutlet opening of the plate sealing means.

In an additional and/or alternative embodiment, the flow of culturemedium is generated by utilizing hydrostatic forces. For example in thatthe level of culture medium in/above the inlet opening is kept higherthan in/above the outlet opening. In an alternative embodiment, a flowof culture medium through the wells being in fluid communication witheach other can be achieved in that the system is placed on a rocker andseesawing the system.

According to the third aspect, the invention provides the use of thesystem as described herein before for propagating cells, preferablyeukaryotic cell, more preferably mammalian cells, and particularlypreferably human cells, most preferably in form of a microtissue orspheroid.

In an embodiment of the third aspect, the use of the system forpropagating cells and/or microtissues comprises the use of the systemfor shipping cells and/or microtissues. The system for propagating cellsand/or microtissues is particularly advantageous for shipping cellsand/or microtissues due to the tight sealing of the wells of themultiwell plate by the corresponding plate sealing means and itsproperties. The use of the system for shipping cells and/or microtissuesprovides better survival rates and better viability of the cells and/ormicrotissues at their destination. Particularly advantageous is that thecells and/or microtissues can be shipped at a temperature of between 18°C. and 27° C., preferably at a temperature of between 21° C. and 25° C.,and that it is not necessary to ship the cells and/or microtissues infrozen or deep frozen conditions for maintaining their survival andviability at a desirable level.

According to further aspects, the invention provides multiwell plates asdescribed herein above as part of the system according to the firstaspect, and plate sealing means as described herein above as part of thesystem according to the first aspect.

1.-20. (canceled)
 21. A system for propagating cells, said systemcomprising a multiwell plate and a plate sealing means for sealing aplurality of wells of the multiwell plate, wherein the plate sealingmeans is made of a resilient elastomer, wherein at least twoneighbouring wells of the multiwell plate are in fluid connection withone another by means of at least one channel connecting said two wells.22. The system according to claim 21, wherein the plate sealing meanscomprises at least two openings arranged at positions of said at leasttwo wells that are in fluid connection with one another, therebyproviding an inlet opening and an outlet opening present in themultiwell plate system.
 23. The system according to claim 22, whereinthe inlet opening and the outlet opening are configured as bores throughthe plate sealing means.
 24. The system according to claim 21, whereinthe plate sealing means comprises a resilient mat made of agas-permeable, antistatic silicone rubber.
 25. The system according toclaim 21, wherein the plate sealing means is a planar mat comprised in alid that further comprises a plate and a circumferential brim.
 26. Thesystem according to claim 25, wherein said circumferential brim haslatches at opposite sides of the lid for engaging in grooves of themultiwell plate, and wherein said plate has through bores which alignwith the wells of the multiwell plate upon being mounted thereto. 27.The system according to claim 26, wherein each of the wells of themultiwell plate comprises a circumferential edge protruding from theupper face of the multiwell plate.
 28. The system according to claim 21any one of the preceding claims, wherein the wells of the multiwellplate comprise a central deepening in the middle of the bottom of thewell, defining a microtissue culture compartment.
 29. The systemaccording to claim 21, wherein at least one of the inlet opening and theoutlet opening is provided with a funnel or chimney.
 30. The systemaccording to claim 21, wherein at least a portion of the wells of themultiwell plate is provided with an ultra-low attachment surface. 31.The system according to claim 21, wherein the resilient elastomer has anoxygen permeability of more than 1*109 cm3*cm/(s*cm2*cmHg).
 32. Thesystem according to claim 21, wherein the resilient elastomer isselected from the group consisting of silicone rubber, dimethylsiliconerubber vinyl methyl siloxane, phenyl vinyl methyl siloxane,fluorosilicone rubber, nitrile rubber and natural rubber.
 33. The systemaccording to claim 21, further comprising means for controlling thetemperature within the wells.
 34. The system according to claim 21,further comprising a temperature data logger as a portable measurementinstrument.
 35. A process for propagating cells, the method comprisingthe steps of: providing a multiwell plate; introducing a suspensioncomprising at least one cell into at least one well of the multiwellplate; sealing at least the well of the multiwell plate bearing thesuspension containing at least one cell with a plate sealing meansdefined in claim
 21. 36. The process according to claim 35, wherein theplate sealing means comprises at least two openings arranged atpositions of said at least two wells that are in fluid connection withone another, thereby providing an inlet opening and an outlet openingpresent in the multiwell plate system.
 37. The process according toclaim 35, further comprising propagating the cells at a temperature ofbetween 4° C. and 37° C.
 38. The method of claim 37 or 38, furthercomprising continuously or discontinuously replacing culture medium. 39.A process of propagating cells comprising providing and propagating saidcells in at least one well of a multiwell plate of a system according toclaim
 21. 40. A pharmacological test assay comprising cultivating cellsin a system according to claim 1 and testing the toxicity of a drug or achemical compound on said cells.